A microscope, in which the illumination beam path and the detection beam path are arranged essentially perpendicular to each other, and in which the specimen is illuminated with a light sheet in the focal plane of the imaging objective, i.e., perpendicular to its optical axis, is designed for the analysis of specimens according to the method of selective plane illumination microscopy (SPIM). In contrast to confocal laser scanning microscopy (LSM), with which a three-dimensional specimen is scanned point by point in different planes with different depths and the obtained image information is subsequently compiled to create a three-dimensional image of the specimen, SPIM technology is based on widefield microscopy and allows the pictorial representation of the specimen on the basis of optical sections through individual planes of the specimen.
The advantages of the SPIM technology consist among other things in the greater speed with which the image information is captured, the lower risk of fading of biological specimens as well as an extended penetration depth of the focus into the specimen.
In principle, the SPIM technology involves the stimulation of fluorophores contained in or incorporated into the specimen with a laser light, which is shaped into a so-called light sheet. In each case, the light sheet is used to illuminate a selected plane in the depth of the specimen and an image of said plane of the specimen is obtained in the form of an optical section using an imaging optical unit. The rapid reciprocating movement of a thin, rotationally symmetric laser beam in the focal plane of the imaging objective is essentially equivalent to said type of stimulus with a static light sheet. In effect, i.e. in the temporal average over the observation period, this results in the shape of a light sheet.
SPIM technology is described for example in Stelzer et al., Optics Letters 31, 1477 (2006), in Stelzer et al., Science 305, 1007 (2004), in DE 102 57 423 A1 and in WO2004/0530558 A1.
The basic design of a SPIM microscope is illustrated in FIG. 1. The light of an illuminating source 1 is shaped to become a light sheet via an illuminating optical unit 2 and guided onto a specimen 3. The specimen and light sheet are located in the focal plane of the imaging objective 4. The optical axis of the imaging objective 4 is perpendicular to the direction from which the specimen 3 is illuminated. Generally, the illuminating optical unit 2 comprises a plurality of optical elements, which collimate the coherent light of the illuminating source 1 and use it to form a light sheet. In the prior art, the illuminating optical unit 2 generally also comprises a cylinder lens, whose flat side is pointing toward the specimen and whose arched side is pointing into the direction of the illuminating source. Several examples of illuminating optical units 2 are explained below, which facilitate the generation of a light sheet with enhanced depth of field and reduced casting of shadows compared to arrangements known from the prior art.